Malignant melanoma of the skin can be highly lethal when the disease is detected in the advanced stages with overall cure rate of <10%. Almost all of malignant melanoma is autotrophic for arginine because these melanoma cells do not express argininosuccinate synthetase (AS), the rate-limiting enzyme for the biosynthesis of arginine from citrulline. Thus, melanoma cells require arginine from extracellular sources for survival. When these melanoma cells are exposed to arginine deiminase (ADI) which degrades arginine to citrulline and ammonia, they become arginine starvation, leading to cell death; while normal cells which express AS are able to survive. ADI is a bacterial enzyme and a pegylated enzyme (ADI- PEG20) has been formulated. Phase I/II clinical trials using ADI-PEG20 for advanced melanoma have produced favorable results. We have learned that re-expression of AS was observed in the ADI-insensitive patients. Consistent with these findings, we found that ADI treatment induces AS expression in some melanoma cells; and the induced AS expression is associated with ADI resistance. Moreover, suppressing induction of AS expression in ADI-treated melanoma cells inhibited the development of resistance. These results suggest that the re-expression of AS is an important mechanism associated with ADI-PEG20 resistance and underscores the importance of understanding the mechanism(s) of AS induction to overcome the development of resistance. We have been studying the mechanism of AS induction in melanoma cells and discovered that the positive regulator c-Myc and the negative regulator HIF-1a are involved. We have also observed that failure of induction of AS expression in some melanoma cells is associated with the inability of c-Myc to interact with the AS promoter. We propose the following four specific aims to further elucidate the regulatory mechanisms of AS expression: Our preliminary results suggest that phosphatidylinositol 3-kinase (PI3K)-AKT pathway may be involved in the regulation of c-Myc- mediated AS induction by ADI-PEG20. We propose in Specific Aim 1 to further elucidate the involvement of this pathway in the induction. Our preliminary results showed that ADI-PEG20 treatment results in downregulation of HIF-1a. We propose in Specific Aim 2 to elucidate the molecular basis underlying the downregulation mechanism. In Specific Aim 3, we propose to investigate the role of c-Myc/HIF-1a in the maintenance of AS expression in the ADI-resistant melanoma cells. We also propose to use small molecules to overcome the resistance through suppression of AS expression. In Specific Aim 4, we propose to investigate whether ADI-PEG20-resistant mechanisms developed in melanoma cells can be validated in tissue specimens derived from melanoma patients treated with ADI-PEG-20. The ultimate goal of this research is to improve therapeutic outcome in advanced melanoma by ADI-PEG20 treatment by fully understanding the molecular basis of AS regulation by arginine availability. PUBLIC HEALTH RELEVANCE: This application investigates the mechanism of resistance to the new treatment modality of human melanoma with arginine deiminase (ADI-PEG20) due to the lack argininosuccinate synthetase (AS) expression in this human malignancy. While ADI- PEG20 has been found to be active in vitro and in phase I/II clinical trials, not all patients respond to arginine deprivation with ADI-PEG20 therapy due to resistance to the drug. We propose approaches to circumvent the development of resistance, thereby to enhance the treatment efficacy of human malignant melanoma with ADI-PEG20.